Premium
Expression and purification of Artogeia rapae lysozyme II cDNA in a baculovirus/insect cell system
Author(s) -
BANG In Seok,
YOE Sung Moon,
KANG Chang Soo
Publication year - 2006
Publication title -
entomological research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.421
H-Index - 20
eISSN - 1748-5967
pISSN - 1738-2297
DOI - 10.1111/j.1748-5967.2006.00019.x
Subject(s) - biology , lysozyme , lytic cycle , sf9 , trichoplusia , microbiology and biotechnology , complementary dna , gene expression , baculoviridae , gene , shuttle vector , recombinant dna , spodoptera , biochemistry , virology , noctuidae , vector (molecular biology) , virus , larva , botany
Previously, the Artogeia rapae lysozyme II (ARLII) gene was isolated and its complete nucleotide sequence was determined by RACE‐PCR from fat body of larvae injected with Escherichia coli . In the present study, the ARLII gene was expressed by using a baculovirus expression vector system (BEVS). The expression level of recombinant (r)ARLII protein was optimized by varying virus titer and time‐course of infection. The optimum protein expression conditions were infection of the cells at a multiplicity of infection of 10, and harvest at 84 h post‐infection. Under these conditions, we estimated the amount of rARLII produced in the BEVS to be 10 mg/mL. rARLII was purified from cell‐conditioned media using cation exchange column and reversed‐phase FPLC methods. Purified rARLII was able to form a clear zone in a lysoplate assay against Micrococcus luteus . The lytic activity was estimated to be 1.53 times higher than that of hen egg white lysozyme (HEWLZ) under the same conditions. The optimum temperature for the lytic activity of the rARLII was 50°C, and its temperature dependency was greater than that of HEWLZ at low temperatures (<65°C).